Every living cell needs energy to survive. This energy is produced, according to a process known as oxidative phosphorylation, in form of the chemical entity adenosine triphosphate (ATP). ##STR1##
The key enzyme in the production of ATP is NADH cytochrome C reductase, also known as Complex I-III. This enzyme reduces cytochrome C by using the reducing agent NADH, the reduced form of nicotinamide adenine dinucleotide. The reduced cytochrome C is then oxidized by the enzyme cytochrome C oxidase (Complex IV) to form water. In other words, the reduced form of NADH, also called Coenzyme I, uses ubiquitous oxygen in the cell to form water and 3 ATP molecules in accordance with the following general reaction scheme: EQU NADH+H.sup.+ +1/2O.sub.2 +3P.sub.i +3ADP.fwdarw.NAD.sup.+ +3ATP+4H.sub.2 O.
Thus, with one NADH molecule, three ATP molecules are obtained having an energy of approximately 21 kilocalories. This process is set forth schematically in FIG. 1, where FeS is Reiske iron sulfur protein; ADP=adenosine diphosphate; and b.sub.562, b.sub.566, c.sub.1, a and a.sub.3 are cytochromes. The enzymes depicted in FIG. 1 are referred to as Complex I (NADH:ubiquinone oxidoreductase); Complex II (succinate dehydrogenase); Complex III (ubiquinone:cytochrome C oxidoreductase); Complex IV (cytochrome C oxidase); and Complex V (ATP synthase). These enzymes, whose energy-related functions occur in the mitochondria of the cell, are assembled from 13 polypeptides coded by the mitochondrial DNA (mtDNA) and approximately 50 polypeptides coded by the nuclear DNA (nDNA). This system of five complexes also constitutes what is referred to as the electron transport chain (ETC), the common pathway for cellular energy metabolism, through which enzyme-catalyzed redox processes achieve electron transfer among critical substrate species.
NADH cytochrome C reductase is the first and key enzyme of this energy producing process. The greater the activity of NADH cytochrome C reductase, the higher the cellular output of energy. Illustratively, the more energy a cell needs, the more NADH it contains. For example, heart cells have 90 .mu.g/g tissue; brain and muscle cells contain 50 .mu.g/g tissue; liver cells contain 40 .mu.g/g; and red blood cells contain 3 .mu.g/g tissue. Thus, the activity of NADH cytochrome C reductase, directly linked to the amount of NADH present in the cell, reflects the energy producing capacity of a cell. Alberts, B., Bray, D., Lewis, J., Raff, H., Roberts, K., and Watson, J. D., "Energy Conversion: Mitochondria and Chloroplasts," in Molecular Biology of the Cell, 3rd Ed., Garland Publishing Inc., pp. 653-720, 1994; Lehninger, A. L., "Vitamins and Coenzymes," in Biochemistry, 2nd Ed., The John Hopkins University School of Medicine, Worth Publishers, Inc., pp. 337-342, 1975.
It has been shown in a variety of diseases (the so-called mitochondrial diseases) that energy production, in particular the activity of NADH cytochrome C reductase (Complex I-III), is decreased. This has been demonstrated not only in brain and muscle tissue, but also in platelets. Cooper, J. M., Mann, V. N., Krige, D., and Schapira, A. H. V., "Human mitochondrial complex I dysfunction," Biochemica et Biophysica Acta 1101,198-203 (1992); Mizuno, Y., et al., "Deficiencies in Complex I Subunits of the Respiratory Chain in Parkinson's Disease," Biochemical and Biophysical Research Communication 163, 1450-1455 (1989); Shoffner, J. M., Wafts, R. L., Juncos, J. L., Torroni, A., and Wallace, D.C., "Mitochondrial Oxidative Phosphorylation Defects in Parkinson's Disease," Ann. Neurol. 30, 332-339 (1991). This has been found both in patients with Parkinson's disease (PD) and in patients with Alzheimer's disease. A further demonstration of the link between cytochrome C reductase activity and disease conditions involves the Parkinson inducing toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a compound that irreversibly inhibits and destroys NADH cytochrome C reductase in certain brain areas causing Parkinsonian-like symptoms. Benecke, R., Strumper, P., and Weiss, H., Brain 1993, Vol. 116, Part 6, pp. 1451-1463. These and other similar findings have significant implications for investigations into the etiology of conditions such as Alzheimer's and Parkinson's diseases.
Another known enzyme toxin is azidothymidine (AZT) which is used in the treatment of AIDS patients. This toxin damages NADH cytochrome C reductase, causing a reduction of energy production in the cell. Dalakas, M. C., lila, I., Pezeshkpour, G. H., Laukaftis, J. P., Cohen, B, and Griffin, J. L. "Mitochondrial myopathy caused by long-term zidovudine therapy," New Engl. J.Med. 322, 1098-1105 (1990). By measuring the activity of NADH cytochrome C reductase in muscle tissue biopsy, it was demonstrated that AZT destroys the enzyme's activity, consequently blocking the energy production of the cells leading to muscle atrophy.
In addition to substances known to have an inhibitory effect on the activity of key enzymes related to cellular processes for the production of energy, there are also substances, such as the reduced form of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), either endogenous or introduced exogenously, that are able to enhance NADH cytochrome C reductase activity and, consequently, cellular energy production. These two co-enzymes, and their pharmaceutically acceptable salts, have been shown to be useful in the treatment of Parkinson's Disease (PD). The effectiveness of these agents for this purpose is disclosed in U.S. Pat. Nos. 4,970,200, 5,019,516, and 5,332,727, the disclosures of which are incorporated herein by reference. In addition, these substances are effective in the treatment of Alzheimer's disease, as disclosed in U.S. Pat. No. 5,444,053, the disclosure of which is also incorporated herein by reference. These substances have also been demonstrated to be effective in supplying additional energy to healthy individuals as disclosed in EP 0 496 479 131.
Assay methods for the determination of NADH cytochrome C reductase activity have been described for many tissues, in particular muscle, liver, brain and heart cells. See, for example, Hatefi, Y. and Stiggall, D. L. (1978b), "Preparation and properties of NADH:cytochrome C oxidoreductase (Complex III)," in Methods in Enzymology, 53, Fleischer, S. and Packer, L., eds, pp 5-10, Academic Press, New York, 1978; Trounce, I., Byrne, E. and Marzuki, S., "Decline in skeletal muscle mitochondrial respiratory chain function: Possible factors in ageing," Lancet 1989, 637-639 (assay of muscle tissue); Yen, T.-C., et al., "Liver mitochondrial respiratory functions decline with age," Biochem. Biophys. Res. Comm. 165, 994-1003 (1989) (assay of liver tissue); Nakagawa-Hattori, Y., et al., "Is Parkinson's disease a mitochondrial disorder?" J. Neurol. Sci. 107, 29-33 (1992) assay of muscle tissue obtained post-mortem); Mizuno, Y., et al, "Effects of 1-methyl-1-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenylpyridinium ion activities of the enzymes in the electron transport system in mouse brain," J. Neurochem. 48, 1787-1793 (1 987) (assay of mouse brain tissue); Reichman, H. et al, "Respiratory chain and mitochondrial deoxyribonucleic acid in blood cells from patients with focal and generalized dystonia," Movement Disorders 9, 597-600 (1994) (assays of platelet homogenate).
Indeed, cofactors in oxidative phosphorylation processes have seen widespread use in analytical procedures due to the extremely high molar absorptivities demonstrated by species such as NADH which exhibits a unique absorption maximum at approximately 340 nm, along with an even stronger maximum at about 270 nm that is shared with the oxidized form of the cofactor, NAD.sup.+. By way of example, the concentration of alcohol in solution can be determined in an enzyme-based assay by adding an excess of NAD.sup.+ and a suitable quantity of the NAD.sup.+ -dependent enzyme, alcohol dehydrogenase. The amount of NADH formed by the enzyme catalyzed reaction, which amount can be easily monitored spectrophotometrically, is stoichiometrically related to the amount of alcohol originally present in solution. The utility of such enzyme-based assays can be further extended through the use of coupled reaction schemes in which the analyte of interest does not directly participate in the enzyme-catalyzed process that gives rise to an analytical signal, but is indirectly linked to such process by a coupled reaction mechanism. However, it should be noted that, despite the use of a common enzyme cofactor in these analytical procedures, all such procedures share a common trait in that they are limited to monitoring processes that are not directly implicated in cellular energy production. As a consequence, such processes are dependent on the addition of both exogenous enzyme and cofactor, such as NADH, in order to generate an analytical signal.
For those demonstrated analytical procedures more directly related to cellular energy processes, a number of specific additional limitations exist. For example, all tissue-based assays require the sampling of tissue through biopsy or other even more invasive surgical procedures. In many cases, the tissue site selected for sampling is so critical physiologically that it can only be sampled post mortem. Assays based on isolation from platelets derived from a blood sample acquired through conventional venipuncture techniques offer advantages over methods that require biopsy of muscle or other tissue. However, such techniques suffer from the serious drawback that observed enzyme activity is dependent on the level of purification of the platelet preparation, introducing a potential source of significant analytical error. Furthermore, platelet separation/purification techniques are inherently complex and time consuming, adding significantly to the procedural overhead of enzyme-based methods from platelets. In addition, all of these enzyme-based assay methods can be further distinguished from the methods of the invention disclosed herein in that they all require the addition of exogenous sources of all cofactors involved in the enzymatic processes. Therefore, none of these methods can be characterized as assessing activity involving endogenous substrate.
In light of these and other significant drawbacks to the currently available assay methods for determination of energy-related enzyme activity, it is recognized that there is a need for an assay method that is simple in both sampling and procedure, as well as one that is efficient and well-suited to routine application. Toward that end, the inventor herein discloses an enzyme-based assay method that can be used with whole blood samples that has utility for assessment of the energy-producing capacity of test subjects, either before and/or after clinical treatments that include the ingestion of exogenous sources of NADH and related compounds.